Increasing interest in the use of macromolecular drug-conjugates is prompting a systematic study of the macromolecule-cell interactions leading to the uptake and intracellular processing of drug-carriers. Three types o carriers containing covalently linked methotrexate will be tested for their growth inhibitory effects on cultured cells. The three carriers, poly(D-lysine), Alpha2-macroglobulin and anti-SSEA-1 monoclonal antibody, are chosen to illustrate and compare the processes on non-receptor-mediated adsorptive endocytosis, receptor-mediated endocytosis and specific interaction of an antibody with its cell surface antigen. Poly(D-lysine) is transported into cells very efficiently and is not degraded inside cells. It is therefore a unique tool to test three different types of drug-carrier linkages, which are cleaved under different conditions, possibly in different cellular compartments. The linkages will be either protease-sensitive, acid-sensitive or sensitive to reducing conditions. The protease-sensitive linkages will include highly digestible proteins and oligopeptides tailored to the specificity of lysosomal enzymes. The acid-sensitive linkage will use cisaconitic acid as a spacer which cleaves spontaneously and releases unaltered drug at pH of 6 and less, i.e. the pH range of lysosomes and prelysosomal vacuoles. Cleavage of the specific linkages will be an absolute prerequisite for drug action in all three cases. Growth inhibition will be correlated tothe amount of carrier internalized, the fraction that is processed and released as small molecular drug, and the specific inhibitory activity of that fraction on dihydrofolate reductase. Comparison of three carriers bearing the same drug-linkage will clarify their respective transport properties and abilities to reach intracellular compartments needed for a specific cleavage. Two carrier-conjugates will be tested on drug resistant CHO cells defective in methotrexate transport to determine which carrier-linkage combination is most effective in overcoming drug resistance. The same carriers will also be used to transport daunomycin into drug-resistant CHO cells and test whether the same approach can be used to overcome a second drug resistance due to another type of membrane defect. The antibody will be used to compare the relative cytotoxicity of a pure immunological, a pure drug-induced and a combined immunological and drug-induced attack on F9 teratocarcinoma cells.